This grant supported the development of two-dimensional (2D) protein crystallography, which led, over the course of 25 years, to electron and X-ray crystal structures of RNA polymerase II (pol II), alone and in the act of transcription. These structures were extended, during the previous project period (2002-2007), to higher complexes with general transcription factors, illuminating the mechanisms of transcription start site selection, promoter melting, abortive initiation, and promoter escape. We now propose the culmination of our efforts, the structure determination of the entire transcription machinery. Biochemical studies of the general transcription factors and of the Mediator of transcriptional regulation during the previous project period have removed the major obstacles to the proposed research. Preliminary crystallographic work has shown the feasibility of accomplishing our goal during the next project period. In parallel with the application of 2D crystallography and other methods to the transcription problem, we have pursued the development of the methodology itself. This work was inspired by an idea for routine high resolution structure determination of large complexes by electron microscopy and single particle analysis. We have proposed to rigidly attach multiple heavy atom clusters to specific sites on the complexes before imaging, and then align the images based on the projected positions of the clusters. This heavy atom electron microscopy, or "HevEM," approach requires perfectly uniform clusters about a nanometer in diameter, as well as chemistries for their conjugation with biological molecules. During the past decade, we have devised procedures for meeting these requirements, and are poised for the first trial of HevEM, as well as its application to the giant transcription protein assemblies of interest. Specific aims for the next project period are as follows: 1. Structure determination of general transcription factor H. 2. Structure determination of pol II - general transcription factor complexes. 3. Structure determination of Mediator and of Mediator - pol II complexes. 4. Development of the HevEM approach. PUBLIC HEALTH RELEVANCE: The significance of the proposed research may be summarized as follows: it will provide the structural information needed to fully understand the fundamental mechanism of transcription;it will establish a structural basis for studies of transcriptional regulation;and it will facilitate the design of new therapies for diseases of aberrant gene regulation in the future.